Skip to main content
SpringerLink
Log in

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry

  • Applied Microbial and Cell Physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The present study reports the disinfection effects of chemically and electrochemically dosed chlorine on two models for typical water-borne bacteria (Escherichia coli and Legionella beliardensis) by plating and flow cytometry (FCM) in combination with different fluorescence dyes. The residual effect on various cell functions, including cultivability, esterase activity, membrane polarization, and integrity, was tested at different free chlorine concentrations. In comparison, chemical disinfection yielded on average 60% more E. coli cells entering the viable but nonculturable (VBNC) state than electrochemical disinfection. Here, VBNC is defined as those cells with intact cell membrane but which cannot be cultured on solid nutrient agar plates. L. beliardensis was about five times more resistant to chlorine disinfection than E. coli. The results also suggested the two methods result in different disinfection mechanisms on L. beliardensis, i.e., chemically dosed chlorine targeted cell membrane integrity before enzyme activity, while electrochemically dosed chlorine acted the other way round. In addition, both bacteria lost the integrity of their cell membranes at three times lower chlorine concentration over a longer contact time (i.e., 40 vs. 10 min) by the chemical method. Our results showed that FCM is an appropriate tool to evaluate the effects of water disinfection and the percentage of cells in VBNC in a matter of hours. Electrochemical disinfection is suggested to be a favorable alternative for chemical disinfection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Allegra S, Berger F, Berthelot P, Grattard F, Pozzetto B, Riffard S (2008) Use of flow cytometry to monitor Legionella viability. Appl Environ Microbiol 74(24):7813–7816

    Article  CAS  Google Scholar 

  • Allen MJ, Edberg SC, Reasoner DJ (2004) Heterotrophic plate count bacteria—what is their significance in drinking water? Int J Food Microbiol 92:265–274

    Article  Google Scholar 

  • Alleron L, Merlet N, Lacombe C, Frere J (2008) Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment. Curr Microbiol 57:497–502

    Article  CAS  Google Scholar 

  • Barbesti S, Citterio S, Labra M, Baroni MD, Neri MG, Sgorbati S (2000) Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria. Cytometry 40(3):214–218

    Article  CAS  Google Scholar 

  • Berney M, Weilenmann H-U, Egli T (2006) Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS). Microbiology 152(6):1719–1729

    Article  CAS  Google Scholar 

  • Berney M, Vital M, Hueshoff I, Weilenmann H-U, Egli T, Hammes F (2008) Rapid, cultivation-independent assessment of microbial viability in drinking water. Water Res 42(14):4010–4018

    Article  CAS  Google Scholar 

  • Bosshard F, Berney M, Scheifele M, Weilenmann H-U, Egli T (2009) Solar disinfection (SODIS) and subsequent dark storage of Salmonella typhimurium and Shigella flexneri monitored by flow cytometry. Microbiology 155(4):1310–1317

    Article  CAS  Google Scholar 

  • Burch JD, Thomas KE (1998) Water disinfection for developing countries and potential for solar thermal pasteurization. Sol Energy 64:87–97

    Article  Google Scholar 

  • Chowdhury S, Champagne P, McLellan PJ (2009) Uncertainty characterization approaches for risk assessment of DBPs in drinking water: a review. J Environ Manage 90(5):1680–1691

    Article  CAS  Google Scholar 

  • Delaedt Y, Daneels A, Declerck P, Behets J, Ryckeboer J, Peters E, Ollevier F (2008) The impact of electrochemical disinfection on Escherichia coli and Legionella pneumophila in tap water. Microbiol Res 163(2):192–199

    Article  CAS  Google Scholar 

  • Devos L, Boon N, Verstraete W (2005) Legionella pneumophila in the environment: the occurrence of a fastidious bacterium in oligotrophic conditions. Rev Environ Sci Biotechnol 4(1):61–74

    Article  CAS  Google Scholar 

  • Dukan S, Levi Y, Touati D (1997) Recovery of culturability of an HOCl-stressed population of Escherichia coli after incubation in phosphate buffer: resuscitation or regrowth? Appl Environ Microbiol 63(11):4204–4209

    CAS  Google Scholar 

  • Feng C, Suzuki K, Zhao S, Sugiura N, Shimada S, Maekawa T (2004) Water disinfection by electrochemical treatment. Bioresour Technol 94(1):21–25

    Article  CAS  Google Scholar 

  • Gião MS, Wilks SA, Azevedo NF, Vieira MJ, Keevil CW (2009) Validation of SYTO 9/Propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila. Microb Ecol 58:56–62

    Article  CAS  Google Scholar 

  • Gopal K, Tripathy SS, Bersillon JL, Dubey SP (2007) Chlorination byproducts, their toxicodynamics and removal from drinking water. J Hazard Mater 140(1-2):1–6

    Article  CAS  Google Scholar 

  • Gregori G, Citterio S, Ghiani A, Labra M, Sgorbati S, Brown S, Denis M (2001) Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl Environ Microbiol 67(10):4662–4670

    Article  CAS  Google Scholar 

  • Hammes F, Berney M, Wang Y, Vital M, Köster O, Egli T (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Res 42:269–277

    Article  CAS  Google Scholar 

  • Hoefel D, Grooby WL, Monis PT, Andrews S, Saint CP (2003) Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques. J Microbiol Methods 55(3):585–597

    Article  Google Scholar 

  • Howard K, Inglis TJJ (2003) The effect of free chlorine on Burkholderia pseudomallei in potable water. Water Res 37(18):4425–4432

    Article  CAS  Google Scholar 

  • Jeong J, Kim JY, Cho M, Choi W, Yoon J (2007) Inactivation of Escherichia coli in the electrochemical disinfection process using a Pt anode. Chemosphere 67:652–659

    Article  CAS  Google Scholar 

  • Keer JT, Birch L (2003) Molecular methods for the assessment of bacterial viability. J Microbiol Methods 53(2):175–183

    Article  CAS  Google Scholar 

  • Kerwick MI, Reddy SM, Chamberlain AHL, Holt DM (2005) Electrochemical disinfection, an environmentally acceptable method of drinking water disinfection? Electrochim Acta 50(25-26):5270–5277

    Article  CAS  Google Scholar 

  • Kiura H, Sano K, Morimatsu S, Nakano T, Morita C, Yamaguchi M, Maeda T, Katsuoka Y (2002) Bactericidal activity of electrolyzed acid water from solution containing sodium chloride at low concentration, in comparison with that at high concentration. J Microbiol Methods 49:285–293

    Article  CAS  Google Scholar 

  • Kraft A, Stadelmann M, Blaschke M, Kreysig D, Sandt B, Schröder F, Rennau J (1999) Electrochemical water disinfection part I: hypochlorite production from very dilute chloride solutions. J Appl Electrochem 29(7):859–866

    Article  Google Scholar 

  • Leclerc H, Moreau A (2002) Microbiological safety of natural mineral water. FEMS Microbiol Rev 26:207–222

    Article  CAS  Google Scholar 

  • Leclerc H, Mossel DAA, Edberg SC, Struijk CB (2001) Advances in the bacteriology of the coliform group: their suitability as markers of microbial water safety. Annu Rev Microbiol 55(1):201–234

    Article  CAS  Google Scholar 

  • Lin YSE, Vidic RD, Stout JE, Mccartney CA, Yu VL (1998) Inactivation of Mycobacterium avium by copper and silver ions. Water Res 32(7):1997–2000

    Article  CAS  Google Scholar 

  • Lisle JT, Pyle BH, McFeters GA (1999) The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7. Lett Appl Microbiol 29:42–47

    Article  CAS  Google Scholar 

  • Lo Presti F, Riffard S, Meugnier H, Reyrolle M, Lasne Y, Grimont P, Grimont F, Benson R, Brenner D, Steigerwalt A, Etienne J, Freney J (2001) Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France. Int J Syst Evol Microbiol 51(6):1949–1957

    CAS  Google Scholar 

  • Lu W, Kiéné L, Lévi Y (1999) Chlorine demand of biofilms in water distribution systems. Water Res 33(3):827–835

    Article  CAS  Google Scholar 

  • Matsunaga T, Nakasono S, Takamuku T, Burgess JG, Nakamura N, Sode K (1992) Disinfection of drinking water by using a novel electrochemical reactor employing carbon-cloth electrodes. Appl Environ Microbiol 58(2):686–689

    CAS  Google Scholar 

  • Meyer B (2003) Approaches to prevention, removal and killing of biofilms. Int Biodeterior Biodegrad 51:249–253

    Article  CAS  Google Scholar 

  • Muraca P, Stout JE, Yu VL (1987) Comparative assessment of chlorine, heat, ozone, and UV light for killing Legionella pneumophila within a model plumbing system. Appl Environ Microbiol 53(2):447–453

    CAS  Google Scholar 

  • Murga R, Forster TS, Brown E, Pruckler JM, Fields BS, Donlan RM (2001) Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147:3121–3126

    CAS  Google Scholar 

  • Nakajima N, Nakano T, Harada F, Taniguchi H, Yokoyama I, Hirose J, Daikoku E, Sano K (2004) Evaluation of disinfective potential of reactivated free chlorine in pooled tap water by electrolysis. J Microbiol Methods 57(2):163–173

    Article  CAS  Google Scholar 

  • Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA (2000) Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Methods 42(1):97–114

    Article  CAS  Google Scholar 

  • Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43:93–100

    Google Scholar 

  • Phe M-H, Dossot M, Guilloteau H, Block J-C (2005) Nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria. Water Res 39(15):3618–3628

    Article  CAS  Google Scholar 

  • Phe MH, Dossot M, Guilloteau H, Block JC (2007) Highly chlorinated Escherichia coli cannot be stained by propidium iodide. Can J Microbiol 53(5):664–670

    Article  CAS  Google Scholar 

  • Robertson BR, Button DK, Koch AL (1998) Determination of the biomass of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry. Appl Environ Microbiol 64:3900–3909

    CAS  Google Scholar 

  • Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346

    Article  CAS  Google Scholar 

  • Temmerman R, Vervaeren H, Noseda B, Boon N, Verstraete W (2006) Necrotrophic growth of Legionella pneumophila. Appl Environ Microbiol 72(6):4323–4328

    Article  CAS  Google Scholar 

  • Venczel L, Arrowood M, Hurd M, Sobsey M (1997) Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine. Appl Environ Microbiol 63(4):1598–1601

    CAS  Google Scholar 

  • Villarino A, Bouvet OMM, Regnault B, Martin-Delautre S, Grimont PAD (2000) Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat–or UV-killed cells. Res Microbiol 151:755–768

    Article  CAS  Google Scholar 

  • Villarino A, Rager MN, Grimont PAD, Bouvet OMM (2003) Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead? Eur J Biochem 270:2689–2695

    Article  CAS  Google Scholar 

  • Vives-Rego J, Lebaron P, Nebe-von Caron G (2000) Current and future applications of flow cytometry in aquatic microbiology. FEMS Microbiol Rev 24(4):429–448

    Article  CAS  Google Scholar 

  • Wang Y, Hammes F, Boon N, Egli T (2007) Quantification of the filterability of freshwater bacteria through 0.45, 0.22, and 0.1 μm pore size filters and shape-dependent enrichment of filterable bacterial communities. Environ Sci Technol 41(20):7080–7086

    Article  CAS  Google Scholar 

  • WHO (2002) The world health report 2002: reducing risks, promoting healthy life. World Health Organization, Geneva

    Google Scholar 

Download references

Acknowledgments

The authors are grateful to the financial support from a research grant from the Flemish Fund for Scientific Research (FWO-Vlaanderen, GP.005.09N), by Ecodis NV, and Chinese National Water Special Project (2008ZX07526-002-01). We highly appreciate the useful advice of Kim Heylen and Kevin Van de Merlen, the technical support from Mike Taghon and Tim Lacoere, and the critical reading of the manuscript by Sam Possemiers, Haydee De Clippeleir, and Liesje De Schamphelaire.

Author information

Authors and Affiliations

  1. Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium

    Yingying Wang, Lieve Claeys, David van der Ha, Willy Verstraete & Nico Boon

  2. Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, NanKai University, Tianjin, 300071, China

    Yingying Wang

Authors
  1. Yingying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lieve Claeys
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David van der Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Willy Verstraete
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nico Boon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nico Boon.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table 1

(DOCX 13 kb)

Supplementary Table 2

(DOCX 12 kb)

Supplementary Table 3

(DOCX 15 kb)

Supplementary Table 4

(DOCX 15 kb)

Supplementary Fig. 1

(DOCX 817 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Claeys, L., van der Ha, D. et al. Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry. Appl Microbiol Biotechnol 87, 331–341 (2010). https://doi.org/10.1007/s00253-010-2526-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2526-2

Keywords

Search

Navigation